Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in the decoding of genetic information in all living cells. Despite their relatively early discovery and recent extensive structural characterization, how they achieve discrimination between closely related amino acid and transfer RNA substrates is under active investigation. Among the fundamental questions for the aaRSs are i) whether there are general mechanistic features shared among enzymes in the same class; ii) the precise step(s) at which aminoacylation is rate limited, and whether amino acid specificity is mediated at that step; iii) how specific recognition elements in transfer RNAs exert their effects; and iv) the specific mechanisms that prevent misactivated and misacylated amino acids from being introduced into cellular proteins. To address these questions, we will make use of rapid quench and stopped flow fluorescence approaches developed during the previous funding period to measure rates of elementary steps in the amino acid activation and aminoacylation reactions, and thereby test the hypothesis that mechanistic features common to the class II aaRS superfamily exist. Our aims include: 1) determining the generality of a substrate-assisted and concerted aminoacylation mechanism discovered in histidyl-tRNA synthetases by investigations of threonyl- and alanyl-tRNA synthetases; 2) clarifying the molecular basis of tRNA recognition by defining the elementary steps at which tRNA identity determinants exert their most profound effects on aminoacylation; 3) correlating elementary steps in the aminoacylation pathway with structural changes in threonyl- and histidyl-tRNA synthetase, making use of intrinsic fluorescence and resonance energy transfer; and 4) determining the mechanism of editing in threonyl- tRNA synthetases by measurement of the rates of elementary steps and the binding thermodynamics of editing analogs. Investigations of aminoacyl-tRNA synthetases draw their relevance from the universal presence of these enzymes in all living systems, and their fundamental role in the evolution and operation of the translational machinery. Differences between prokaryotic and eukaryotic enzymes have been exploited in the development of new antibiotics, as well as the incorporation of unnatural amino acids into proteins. The histidyl-tRNA synthetase family is composed of three subgroups with regulatory functions, and the GCN2 subfamily is emerging as a novel regulatory protein with a role in brain function. [unreadable] [unreadable]